SUIT-009 AmR mt D021

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SUIT-009 AmR mt D021

Description

SUIT-009

Abbreviation: H2O2 mtprep

Reference: B: short protocol for H2O2 (AmR) in mitochondrial preparations; mt: isolated mitochondria, tissue homogenate and permeabilized cells - SUIT-009short protocol for H2O2 (AmR) for isolated mitochondria, tissue homogenate and permeabilized cells

SUIT number: D021_1S;2D;3P;4Rot;5Ama

O2k-Application: AmR


SUIT-009 is a short protocol for simultaneous determination of O2 flux and the rate of H2O2 production (H2O2 flux) in mitochondrial preparations such as isolated mitochondria, tissue homogenate (except of liver) and permeabilized cells (already permeabilized when they are added to the chamber). Succinate (S) supports the reverse electron transfer (RET)-related H2O2 production in the LEAK state. Pyruvate (P) supports NADH-pathway (N), and usually in the presence of ADP it does not increase further the H2O2 flux. Antimycin A (Ama) inhibits Complex III (CIII) at the Qo level and could increase the H2O2 flux. The sensitivity of the Amplex UltraRed® assay (for determining H2O2 production) changes over the experimental time and upon addition of chemicals. To correct H2O2 flux for the sensitivity changes several H2O2 calibration steps are done during the experiment. Since the H2O2 flux measured in MiR05-Kit showed a linear correlation with the oxygen concentration, it is recommended to change the oxygen level during the experiment.

Communicated by Komlodi T, Gnaiger E (last update 2020-06-12)

D021 O2 trace.png

D021 AmR trace.png

MitoPedia: SUIT

Steps and respiratory states

1S;2D;3P;4Rot;5Ama.png


Step State Pathway Q-junction Comment - Events (E) and Marks (M)
0DTPA
  • DTPA is an iron chelator, which decreases the chemical fluorescence background created by the Amplex UltraRed assay. Administration of DTPA into the O2k-chamber is recommended before all other chemicals because the iron chelation capacity of the compound is time-dependent (approx. 10-15 min). However, the experiments can be carried out in the absence of DTPA.
0SOD
  • SOD or superoxide dismutase converts the anion superoxide released by the mitochondria into H2O2, making it accessible to the Amplex UltraRed assay.
0HRP
  • HRP or horseradish peroxidase catalyses the conversion of Amplex UltraRed and H2O2 towards the fluorescent resorufin.
0AmR
Step State Pathway Q-junction Comment - Events (E) and Marks (M)
1S SL(n) S CII 1S
2D SP S CII 1S;2D
3P NSP NS CI&II 1S;2D;3P
  • Respiratory stimulation by simultaneous action of type N substrates & succinate, with convergent electron flow in the NS-pathway for reconstitution of TCA cycle function.
  • OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
4Rot SP S CII 1S;2D;3P;4Rot
5Ama ROX 1S;2D;3P;4Rot;5Ama
  • Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated after addition of antimycin A (inhibitor of CIII). Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration (mt).


Questions.jpg


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Strengths and limitations

  • This is a protocol designed to analyze:
1. the S- and NS-pathway-linked respiration and H2O2 flux.
2. any Complex I defect, which would lead to a decrease in N-linked respiration and in reverse electron transfer-supported H2O2 flux (RET).
3. any Complex II defect, which would result in an altered S-linked respiration and H2O2 flux.
+ This protocol is designed to evaluate RET in the S-linked pathway in the LEAK state, which is decreased by ADP due to reduction of the mt-membrane potential.
+ Short protocol.
- CIV activity and cytochrome c test cannot be performed together with the AmR fluorescence assay.
- We do not generally recommend the addition of already permeabilized cells into the chamber. Instead, we recommend performing the permeabilization of the plasma membrane in the chambers with titration of digitonin.

Compare SUIT protocols

  • SUIT-006 AmR mt D048 to investigate the dependence of H2O2 flux on the mt-membrane potential on the N-control state in isolated mitochondria, tissue homogenate and permeabilized cells (already permeabilized when they are added to the chamber).
  • SUIT-018 are short protocols to study the oxygen dependence of O2 flux and H2O2 production on isolated mitochondria or tissue homogenate.

Chemicals and syringes

Step Chemical(s) and link(s) Comments
H2O2 Hydrogen Peroxide (H2O2)
Step Chemical(s) and link(s) Comments
0DTPA DTPA This step can be skipped
0SOD Superoxide Dismutase (SOD)
0HRP Horseradish peroxidase (HRP)
0AmR Amplex UltraRed (AmR)
Step Chemical(s) and link(s) Comments
1S Succinate (S)
2D ADP (D)
3P Pyruvate (P)
4Rot Rotenone (Rot)
5Ama Antimycin A (Ama)
Suggested stock concentrations are shown in the specific DL-Protocol.

References

 YearReferenceOrganismTissue;cell
MiPNet24.10 H2O2 flux analysis2019-08-05
O2k-Protocols
Hydrogen peroxide flux analysis using Amplex UltraRed assay in MiR05-Kit with DatLab 7.4
MiPNet20.14 AmplexRed H2O2-production2019-06-24
O2k-Protocols
O2k-FluoRespirometry: HRR and simultaneous determination of H2O2 production with Amplex UltraRed.
MouseHeart
Komlodi 2018 J Bioenerg Biomembr2018Komlódi T, Geibl FF, Sassani M, Ambrus A, Tretter L (2018) Membrane potential and delta pH dependency of reverse electron transport-associated hydrogen peroxide production in brain and heart mitochondria. J Bioenerg Biomembr 10.1007/s10863-018-9766-8.Guinea pigHeart
Nervous system
Komlodi 2018 Methods Mol Biol2018Komlodi T, Sobotka O, Krumschnabel G, Bezuidenhout N, Hiller E, Doerrier C, Gnaiger E (2018) Comparison of mitochondrial incubation media for measurement of respiration and hydrogen peroxide production. Methods Mol Biol 1782:137-55.Human
Mouse
Skeletal muscle
HEK
Makrecka-Kuka 2015 Biomolecules2015Makrecka-Kuka M, Krumschnabel G, Gnaiger E (2015) High-resolution respirometry for simultaneous measurement of oxygen and hydrogen peroxide fluxes in permeabilized cells, tissue homogenate and isolated mitochondria. Biomolecules 5:1319-38.Human
Mouse
Heart
Nervous system
HEK
Krumschnabel 2015 Methods Mol Biol2015
O2k-Protocols contents
Krumschnabel G, Fontana-Ayoub M, Sumbalova Z, Heidler J, Gauper K, Fasching M, Gnaiger E (2015) Simultaneous high-resolution measurement of mitochondrial respiration and hydrogen peroxide production. Methods Mol Biol 1264:245-61.
MouseNervous system


 YearReferenceOrganismTissue;cell
Chang 2018 Life Sciences Meeting 2018 Innsbruck AT2018pH dependence of mitochondrial respiration and H2O2 production in oral cancer cells – a pilot study.HumanHEK
Kidney


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